Abstract T cells are central effector cells of protective anti-tumor immunity, but little is currently known about these important immune cells in human GBM. We have generated single-cell RNA-seq data on tumor-infiltrating T cell populations from GBM patients at initial diagnosis or relapse. We used these full-length RNA-seq data to identify clonally expanded T cell populations based on their TCR? and ? chain sequences and then examined which genes were overexpressed by such expanded T cells. This analysis highlighted the KLRB1 gene which encodes the CD161 receptor that was previously shown to inhibit NK cell-mediated cytotoxicity. The CD161 ligand, CLEC2D, is expressed at the cell surface of human GBM cells. We therefore hypothesize that the CD161 ? CLEC2D pathway inhibits the anti-tumor function of both CD8 and CD4 effector T cell populations in GBM. Preliminary data show that inactivation of the KLRB1 gene in primary human T cells greatly enhances their effector function in a humanized mouse model of GBM. Our preliminary data also demonstrate that several other inhibitory receptors are expressed by substantial populations of GBM-infiltrating T cells, including CD96 and two prostaglandin E2 receptors (EP2 and EP4). Aim 1 will focus on the analysis of tumor-infiltrating T cells in GBM patients enrolled in the phase 1b NeoVax plus PD-1 antibody trial described in Project 1. These studies will primarily focus on the expression of inhibitory receptors by T cells and their ligands by tumor cells and myeloid cells. Expression of inhibitory receptors and their ligands will be examined by 16-color spectral flow cytometry and single-cell RNA-seq (in collaboration with Cores 1 and 2), with an emphasis on CD161, PD-1, CD96 and prostaglandin E2 receptors. We will investigate paired tumor samples from the same patient obtained at initial surgery and relapse in order to determine how expression of these inhibitory receptors and their ligands changes following immunotherapy with NeoVax plus PD-1 antibody. In collaboration with Project 1, we will also examine the spatial distribution of T cells that express CD161 and other inhibitory receptors. Aim 2 will investigate the therapeutic significance of the CD161 ? CLEC2D pathway. We will first use a genetic approach to study this inhibitory receptor by inactivating the KLRB1 gene in primary T cells. Blocking mAbs specific for human CD161 will also be used to examine the therapeutic potential of these findings. We will also examine combination therapies (collaboration with Projects 2, 4 and Core 3) involving the inhibitory receptors identified by single-cell RNA-seq in human GBM infiltrating T cells, with a particular focus on CD161, PD-1, CD96 and the prostaglandin E2 receptors. These studies will significantly advance our understanding of T cell function in GBM and characterize important inhibitory receptor ? ligand interactions that constrain effector T cell function.